This invention relates to arrayable thermal or calorimetric measurement systems allowing precise, reproducible calorimetric assays for physical characterization of biochemical products, as well as for use in pharmaceutical and biotechnology product development. Specifically, this invention uses a novel calorimetric method and apparatus employing base thermopiles in thermal communication with a plurality of samples, to provide thermal signatures, such as changes in specific heat capacity or capacitance, to differentiate or rank biological or chemical samples. This allows systems having arrays of cells, and using facile automated handling, such as by use of disposable tubes, microtiter plates, robotic transports, and the like to perform thermophysical assays, such as for sample screening or to characterize chemical or biological activity.
Biological calorimetry is a well known technique used as a marker to identify systems, or to detect phase transformations or reactions such as binding of ligands to proteins (see A. E. Beezer, Biological Calorimetry, (copyright) 1980, Academic Press, New York). Useful measurements for calorimetric assays include the derivative with respect to temperature of a unique state function, the enthalpy H, yielding the heat capacity
Cp=d H(T)p/dTxe2x80x83xe2x80x83(1)
at constant pressure p. Direct calorimetric measurements allow determination of the heat capacity Cp of a sample, measured in kJ/K or similar units. By determining heat capacity in a selected temperature region, one can determine the enthalpy H, and in turn other state functions such as the sample entropy S, and the Gibbs Function G. See P. W. Atkins, Physical Chemistry, 6th Ed., W. H. Freeman and Company, New York (copyright) 1997, ISBN 0716728710. It is useful to note that heat capacity is an intensive property, independent of the quantity or shape of the substance under consideration. The corresponding extensive property is known as heat capacitance, in analogy with electrical capacitance, and is a function of the quantity of substance involved.
Many processes involving biopolymers and proteins take place with detectable changes in apparent heat capacities of the reacting species. A molecule or biochemical system that has or obtains many translational, rotational, and vibrational degrees of freedom will have a high heat capacity Cp while a simpler (e.g., folded protein) system will have a lower heat capacity. Determining the heat capacity Cp therefore yields an important thermophysical property, and can be used for assays and structure determinations for solutions, proteins, and biological samples. Such assays and structure determinations can be useful for sample screening and biochemical product synthesis.
Six possible sources of large heat capacity (and entropy) changes have been identified for processes involving proteins (Julian M. Sturtevant, Proc. Natl. Acad. Sci. USA, Vol 74, No. 6, pp.2236-2240, June 1977). Protein structure changes such as unfolding can produce large changes in heat capacity, such as unfolding of xcex1-Chymotrypsin, which yields a change in heat capacity Cp of +3080 cal/K/mol at neutral pH. Processes include hydrophobic effects, where nonpolar groups raise the heat capacities of solutes in aqueous solutions; electrostatic effects, where creation of positive and negative charges in aqueous solutions leads to a negative change in heat capacity; breaking of hydrogen bonds with increasing temperature, where heat capacity increases; intramolecular vibrations, affected by chemical changes such as unfolding or ligand binding, where an increase in the number of easily excitable internal vibrational modes results in heat capacity increases; or changes in equilibria, where an actual shift with temperature of an equilibrium between two or more states will appear experimentally as a contribution to the heat capacity.
Current views about protein-ligand interactions state that electrostatic forces drive the binding of charged species and that burial of hydrophobic and polar surfaces influences or controls the heat capacity changes associated with the reaction. However, concerning interactions of a protein with a monovalent cation where electrostatic forces are expected to be significant due to the ionic nature of the ligand, heat capacity changes are expected to be small due to the small surface area involved in the protein-ligand recognition event. It has been found, however, that with the physiologically important interaction of Na+ with thrombin, binding is characterized by a modest dependence on ionic strength, but a large negative heat capacity change of xe2x88x921.1 xc2x10.1 kcal/mol/K (see Guinto, Cera, Biochemistry, Vol 35, No 27, pp. 880-8804). It is proposed that this change is linked to electrostatic effects can reveal a binding or folding event where water molecules are buried, resulting in significant heat capacity changes independent of changes in exposed hydrophobic surface or coupled conformational transitions (rotations about a single chemical bond). Generally, monovalent cation binding to proteins is a widespread phenomenon and can play an important role in enhancing catalytic activity of enzymes. Potassium ion binding to proteins is typically accomplished mainly through two mechanisms. In one mechanism, K+ forms a ternary complex with the enzyme and substrate (e.g., ATPases). In another mechanism, as seen in pyruvate kinase, K+ binds to a distinct site and influences the activity of the enzyme in an allosteric fashion, thereby causing a change in the function of the enzyme. Sodium binding can also be important, for example, Na+ activated enzymes are involved in blood coagulation and complement cascades.
Solvation of charged and polar groups is typically accompanied by a negative heat capacity change which is small and only known for simple molecules. Heat capacity of water molecules sequestered in the interior of a protein is significantly lower than in bulk water, because of reduced mobility and more ordered structure. Burial of water molecules linked to ligand binding or protein folding can result in large negative heat capacity changes, which can be detected in an assay using the disclosed invention.
As another example, the binding of L-aribinose and D-galactose to the L-aribinose-binding protein of Escherichia coli has been studied by isothermal and scanning calorimetry (see Fukada, Sturtevant, Journal of Biological Chemistry, Vol. 258, No. 21 pp. 13193-13198, 1983). It is found that the binding reaction with arabinose is characterized by an enthalpy change of xe2x88x9215.3 kcal/mol, with a large decrease in apparent heat capacity of xe2x88x920.44 kcal/mol/K. However, determination methods used are painstaking, typically done with two samples at a time (such as an experimental sample and a control or reference sample), and involve elaborate experimental and chemical procedures.
Thermophysical assays have the advantage of not requiring the use of any external or added agents, such as fluorescent or radioactive tags, which can cause damaging or unknown perturbations on a biochemical system. The heat capacity Cp is also an equilibrium property that can be used to great advantage. Thus, while a small molecule ligand that does not bind to a protein should have a negligible effect on the heat capacity of a system, a small molecule that does bind usually causes a permanent change in molecular degrees of freedom, and hence the heat capacity Cp of the system. Many measurements can thus be made at leisure, after the binding event.
Thermophysical assays can also elucidate cellular processes. One especially important cellular protein, calmodulin (CAM), appears to be at the junction of many signal transduction processes. CAM appears to be able to modulate many distinct processes because it can exist in a large ensemble of different structural states, presenting a corresponding large ensemble of chemical interaction sites. As with most molecular structures, each distinct state is likely to have a characteristic heat capacity Cp Elucidating the network of signal pathways through CAM via heat capacity characterizations would likely have enormous implications for the pharmaceutical industry. Another area where thermophysical techniques can give information about cellular mechanisms is the possibility of elucidating the action of rapamycin, which has been observed to cause immunosuppression by interfering with a calcineurin pathway. Calcineurin is a CAM-dependent phosphotase.
Presently, direct calorimetric observations are made using painstaking physical methods of thermal analysis to determine enthalpy changes in a sample under study. In one known method, isothermal titration calorimetry, a first solution containing a ligand is incrementally titrated (using a gear-driven plunger that actuates a syringe) into a second solution containing a macromolecule or other receptor. Heat released or absorbed upon interaction of the two solutions is measured and plotted as a function of time. As the macromolecule becomes saturated with ligand, the binding heat signal decreases until full saturation, where only background heat from dilution of the ligand is detected. See Nature, Vol. 384, pp. 491-492, Dec. 5, 1996; also Freire, E., Mayorga, O. L., Straume, M., Analytical Chemistry, Vol. 62, pp. 950A-959A (1990). A serious drawback to this technique is the high relative quantities of reactant-containing solution required, and the titration or metering hardware needed makes arrayable assays difficult, particularly if the samples are later to become part of a further synthesis or the making of a dosage form.
Two other typical known techniques used are Differential Scanning Calorimetry (DSC) and Differential Thermal Analysis ( DTA). In DTA, a single heater imparts thermal energy to both a sample pan and a reference or control pan at a constant predetermined rate, such as can be measured in calories/hour, joules/sec, or similar units. When a thermodynamic change occurs in the sample (e.g., a binding event), or if there is an inherent difference in heat capacitances between the sample and reference, there is a resultant temperature difference xcex94T, which is proportional to the enthalpy change xcex94H, the heat capacity Cp, and the total thermal resistance R. This thermal resistance is common to both DTA and DSC, and has two components, R0 and RS:
R=R0+RSxe2x80x83xe2x80x83(2)
R0 is the inherent thermal resistance of the instrument, due to the thermal separation of the sample, heater and thermometer or temperature measuring device(s). RS is the thermal resistance of the sample itself, and includes other peripheral factors like the imperfect thermal contact between the sample and its container or pan. Because thermal resistance RS is a property of the sample, in DTA, RS cannot be determined through a calibration with the reference. As a result, a plot of xcex94T is dependent on the sample, making calculation of heat capacitance and heat capacity Cp very difficult.
Differential scanning calorimetry (DSC) is a well-known method used worldwide to study energetic changes in solid or liquid samples with high precision and accuracy. DSC has been used to determine affinity of a set of azobenzene ligands for streptavidin (see Weber, P. et al., Journal of the American Chemical Society, Vol. 16, pp. 2717-2724, 1994). In DSC, a power compensation technique is used via two separate heaters, and two control loops that operate to keep the sample and reference at the same temperature. A first control loop regulates the average heating rate of both the sample and reference, and a second control loop provides a differential thermal power input q to eliminate any temperature difference between the sample and reference due to an inherent thermophysical difference in the sample or due to a thermodynamic change, such as a heat of reaction. Associated with the second control loop, the rate of this differential thermal power input q to the two heaters, dq/dt, (the derivative of q with respect to time t) is recorded and can be related to the heat flux by
dH/dt=xe2x88x92dq/dt+(CSxe2x88x92CR)dT/dtxe2x88x92RCSd2q/dt2xe2x80x83xe2x80x83(3)
With a constant ambient pressure to which the sample and reference are subjected, dH/dt is simply the rate of absorption of heat per unit time; dq/dt is equal to the differential power input; CS and CR are the heat capacities of the sample and reference, respectively; T is temperature in Kelvin; and R is the thermal resistance given above. The second term on the right is negligible provided the sample and reference have comparable heat capacities; the last term on the right results from thermal lag and can be minimized by making the sample and reference as small as possible, reducing RS. This reduces thermal resistance R from above equation (2) to an acceptable level. Under these conditions, integration of the data curve will yield, to a good approximation,
Cp=d H(T)p/dTxcx9cxe2x88x92dq/dtxe2x80x83xe2x80x83(4)
DSC techniques and numerical methods are known in the art. See J. L. Naughton, C. T. Mortimer, Thermochemistry and Thermodynamics, ed. H. A. Skinner, Butterworths, London, Vol. 10, (copyright) 1975. For larger sample sizes, the above thermal lag can be corrected by use of the Tian equation and conversion to an alternate excess heat capacity versus temperature scan (see Frederick P. Schwartz, Biochemistry, Vol. 27, pp. 8429-8436).
Both the DTA and DSC techniques have serious limitations. One limitation involves the high accuracy required for DSC and DTA measurements. For macromolecular interactions, for example, when active concentrations fall below 0.3 percent, the effects on the heat capacity Cp become negligible, making only a contribution of heat capacity of 1/10 percent (10xe2x88x923) of the total. Determining differences in heat capacity Cp in solutions containing macromolecules in such a solution requires an exceptionally accurate calorimetric measurement. Newer scanning microcalorimetry techniques are often used to determine relative heat capacity Cp within 0.002 percent. However, these thermal techniques are limited by the small sample quantities available and the relatively high amount of experimental effort required for accurate measurements. Homogeneous preparations of high quality biological specimens cannot always be obtained.
As a result, heat capacity for systems of biological interest is generally measured using xe2x80x9cvan""t Hoff analysisxe2x80x9d and not direct calorimetric determinations, which cannot be carried out in a system where the ligand binds in the millimolar range (Biochemistry, Vol. 35, No. 27, pp. 8800-8804, 1996). This type of analysis arises from solving the Gibbs-Helmholtz equation, and plotting 1n K versus 1/T, where K is the relevant reaction rate or equilibrium constant for the reaction under study, and assuming that the heat capacity changes are constant over a temperature range of interest, e.g., 5xc2x0 C. to 45xc2x0 C. The analysis involves determining the reaction rate K in non-calorimetric ways that make arrayable assays difficult.
There is therefore a need for a system and method allowing micromethods that facilitate arrayable thermal assays. Such a system should be able to provide information about the relative binding affinities of different ligands for a receptor protein, for many samples simultaneously. A calorimetric assay system is also needed to facilitate screening of combinatorial libraries, which are collections of chemical or biochemical compounds synthesized by combining chemical xe2x80x9cbuilding blocksxe2x80x9d or groups as reagents, typically in a combinatorial or quasi-combinatorial manner. An enormous number of compounds can be created, with theoretically distinct compounds numbering in the millions or billions (109). Combinatorial libraries can be screened, for example, by examining the extent of binding of a reagent with a target molecule of interest. A filamentous phage display peptide library (which is a form of combinatorial library created by recombinant technology) can be screened for binding to a biotinylated antibody, or other receptor. Often, library screening techniques require the use of chemical labels or tags. There is a real need for acquiring relative binding affinities for a large number of samples in a short time without the use of chemical markers. For isothermal titration calorimetry, Differential Scanning Calorimetry (DSC) and Differential Thermal Analysis (DTA), the approach is time consuming. Three thermal scans per day are routine, and relatively large sample sizes limit productivity.
Generally, as sample size gets smaller, the surface/volume ratios increase, allowing a lower thermal resistance RS, and the smaller mass facilitates timely ramping of temperature with minimum error. It is desirable to have a calorimetric system allowing arrayable measurements, and yet minimal and constant heat exchange properties which do not vary from assay to assay. A high assay throughput is desired, consistent with desired accuracy, and the need for consistent, reproducible results for each assay. A desirable system should be able to detect denaturation, ligand binding, and other changes with active molecules in the millimolar range or less.
However, thermophysical assay apparatuses meeting these requirements are quite difficult and subject to noise and error. A high degree of temperature measurement sensitivity is required, with minimum detection thresholds of 10 microKelvin or less, and even less allowable temperature error.
A number of problems make arrayable thermophysical assays difficult. With any temperature driven assay system, thermal leakage due to conduction, convection and thermal (T4) radiation can skew results, sometimes unpredictably. Thermal leakage includes both leakage or coupling in the ambient space around samples to be tested, and also cross-talk, where thermal energy intended for one sample is coupled to another, or where one sample xe2x80x9cwarmsxe2x80x9d another.
Thermal gradients are also problematic, because it is difficult to maintain across an array temperature uniformity to the thresholds required, particularly when a system is temperature driven using resistive tapes or other discrete heat sources. Close packing of samples in an array can minimize the effect of thermal gradients, but one then risks introducing another large source of error, because closely packed samples increase the effect of cross-talk. Tight temperature regulation whereby samples are temperature-driven to be the same temperature during the course of testing can reduce the effect of cross-talk, as discussed below.
The adequacy of thermal contact between samples and their respective containers, and test cells or heat input/temperature sensing devices also introduces a source of error, although usually one can in large part compensate for differences in thermal resistance R and conductance by prior calibration and the use of reference samples or cells, containing substances of known composition and thermal characteristics (e.g., lab grade pure deionized water).
With use of electrical sensing/control devices for temperature sensing down to under 10 micro Kelvin sensitivity, and the use of active devices for heat input, electrical noise and drift can cause problems, particularly when amplifiers are presented with absolute DC signals that tend to cause drift and other errors.
It is one object of this invention to permit arrayable thermal assays using thermoelectric devices, where reliable relationships between electric potential differences and temperature differences are exploited in solid or liquid materials, with switching made possible between active (heat input) and control (sensing) modes. It is another object to provide arrayable assay techniques that measure either heat capacity, and/or heat gained or lost in real time, inside samples where active macromolecules or other reactants are only in the millimolar range or less. It is yet further an object of the invention to allow simplified feedback control of an arrayable thermal assay, and to have need for fewer reference samples of known composition to reduce errors, using dual data feeds from thermoelectric devices. It is yet a further object to make thermal assays arrayable by compensating for, and avoiding, thermal (temperature) gradients across an array, using thermoelectric devices. It is yet a further object to reduce electrical noise and measurement error in the temperature sensing functions of such a thermophysical assay. Other objects will become apparent upon reading of the specification.
Using the teachings of this invention, one can measure heat capacities and generate thermal signatures as a function of temperature and time for thermal assay arrays, and compensate for or avoid problems associated with thermal temperature gradients across the array that can make accurate measurements difficult. One can also use disposable sample containers and make measurements quickly while driving the assay at the same time. The invention includes a method for performing thermal assays (e.g., thermal signature assays) on a two or more samples in an array, using active/control base thermopiles, the method comprising one or more of the following:
(a) performing a heat transfer to the two or more samples in each of a two or more containers, using a base thermopile in thermal communication with the two or more containers, or using a local heater in thermal communication with the two or more containers;
(b) determining a total heat transferred to the samples by the base thermopile in step (a);
(c) sensing in real time a temperature difference between a first sample and a second sample of the two or more samples of step (a), wherein, in one embodiment, the sensing in real time is performed by a differential thermopile or an individual base thermopile;
(d) performing an additional heat transfer adjustment on the basis of the temperature difference, the additional heat transfer adjustment sized and targeted to at least one of the first and second samples to drive the temperature difference toward zero between the first and second samples;
(e) determining the size of the additional heat transfer adjustment during step (d) for each of the first and second samples;
(f) comparing the size of the additional heat transfer adjustment during step (e) for each of the first and second samples, and ranking the first and second samples according to their respective additional heat transfer adjustments during step (e);
(g) determining heat capacitance for each of the first and second samples using any of: the total heat transferred in step (b), and the additional heat transfer adjustment during step (d);
(h) calculating from the heat capacitances determined in step (g), the heat capacity (Cp) for each of the first and second samples; and
(i) comparing the size of the additional heat transfer adjustment during step (e) for each of the first and second samples, and generating therefrom a thermal profile (e.g., signature) from successive applications of the method during a ramp in temperature of at least one of the first and second samples.
The base thermopile can be electrically driven via the Peltier effect, and the heat transfer using the base thermopile can occur with respect to an isothermal plate in thermal communication with a junction of the base thermopile. The isothermal plate can also be in thermal communication with a strip heater, and the method can additionally comprise ramping the temperature of the isothermal plate using the strip heater.
Optionally, step (a) can comprise transferring heat to the sample using an individual base thermopile in thermal communication with fewer than all of the samples in the two or more samples in the array.
The invention includes this method wherein the heat transfer adjustment comprises applying heat directly using a local heater. Such a local heater can be selected from the group consisting of a resistive device; a non-ohmic device; a device utilizing electromagnetic induction as an energy transfer method; a device operating primarily by light emission; a sonic device; a speaker; a device using combustion-based heating; a device that mediates exposure to one or more heat sinks, using at least one barrier; a chemical device utilizing a phase transformation of a substance for heating; a mechanical system to convert mechanical energy to heat energy; and a device using Bernoulli flow of a carrier medium to transfer heat.
Step (d) can additionally comprise calculating total net heat transferred (or a thermal denaturation curve) to characterize denaturation of a protein or nucleic acid; a binding event; a chemical reaction; a phase transformation, or a change in the basal or metabolic state of a cell, cellular components, or tissue. The total heat transferred in step (b) can be determined by monitoring the control voltage of the base thermopile. Step (a) can also comprise applying an AC waveform to drive the base thermopile, thereby creating a time varying rate of heat transfer by the base thermopile.
Step (a) can comprise applying an AC waveform to drive the local heater, thereby creating a time varying rate of heat transfer by the local heater. Also, the heat transfer using the local heater can occur with respect to an isothermal plate in thermal communication with the local heater. An isothermal plate can be in thermal communication with a strip heater, and the method additionally can comprise ramping the temperature of the isothermal plate using the strip heater.
The invention can also include teachings relating to an arrayable thermal assay apparatus using active/control base thermopiles for performing thermal assays on a two or more samples in an array, the apparatus comprising one or more of the following:
(1) a base thermopile (BT) in thermal contact with a two or more containers, in the array, each of the two or more containers retaining one of the two or more samples;
(2) a differential thermopile with a first opposed thermal junction in thermal contact with a first container, and a second opposed thermal junction in thermal contact with a second container;
(3) first and second local heaters in individual thermal contact with the first and second containers, respectively, wherein the base thermopile is configured and driven to perform a heat transfer to the two or more containers, and the differential thermopile is configured and monitored to sense a relative temperature difference between the first and the second containers; and wherein the first and second local heaters are configured and driven to perform an additional heat transfer adjustment on the basis of the relative temperature difference. The two or more containers can comprise wells in a microtiter plate. Also, the base thermopile can be an individual base thermopile in thermal communication with fewer than all of the samples in the two or more samples in the array. The individual base thermopile can be configured and monitored to sense a relative temperature difference between the first and the second containers.
Optionally, the first and second local heaters are individually selected from a heater group consisting of a resistive device; a non-ohmic device; a device utilizing electromagnetic induction as an energy transfer method; a device operating primarily by light emission; a sonic device; a speaker; a device using combustion-based heating; a device that mediates exposure to one or more heat sinks, using at least one barrier; a chemical device utilizing a phase transformation of a substance for heating; a mechanical system to convert mechanical energy to heat energy; a device using Bernoulli flow of a carrier medium to transfer heat.
Optionally, one can also add a correlated double sampling system to reduce noise and drift from calorimetric determinations, the correlated double sampling system comprising:
an input amplifier connected to provide gain for a thermopile output signal from at least one thermopile selected from the group consisting of a base thermopile and a differential thermopile;
an AC coupled amplifier connected to provide gain to an input amplifier output signal from the input amplifier;
a sample and hold circuit having a sample and hold input connected to an output signal of the AC coupled amplifier;
a chopper circuit to cycle an input amplifier input signal to the input amplifier between the thermopile output signal and a reference voltage, and to also cycle synchronously the sample and hold input between an AC coupled amplifier output signal from the AC coupled amplifier and the reference voltage.